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The development of insulin resistance and type 2 diabetes (T2D) involves a complex array of metabolic defects in skeletal muscle. An in vitro cell culture system excludes the acute effects of external systemic factors existing in vivo. Thus, we aimed to determine whether intrinsic differences in the protein profile exist in cultured myotubes derived from T2D versus normal glucose tolerant (NGT) healthy people. Applying two dimensional difference gel electrophoresis technology (2-D DIGE), the abundance of 47 proteins differed in myotubes derived from T2D patients versus NGT donors. Proteins involved in fatty acid and amino acid metabolism, TCA cycle, mitochondrial function, mRNA processing, DNA repair and cell survival showed higher abundance, while proteins associated with redox signaling (PARK7; Parkinson disease 7), glutathione metabolism (glutathione S-transferase, GST, isoforms T1, P1 and M2), and protein dynamics (heat shock protein, HSP, isoform B1 and 90A) showed reduced abundance in myotubes derived from T2D versus NGT donors. Consistent with our proteome analysis results, the level of total glutathione was reduced in myotubes obtained from T2D versus NGT donors. Taken together, our data provide evidence for intrinsic differences in the profile of proteins involved in energy metabolism, cellular oxidative stress, protein dynamics and gene regulation in myotubes derived from T2D patients. These differences thereby suggest a genetic or epigenetic influence on protein content level, which can be further investigated to understand the molecular underpinnings of T2D progression and lead to new therapeutic approaches.

DJ-1 is an oxidation sensitive protein encoded by the PARK7 gene. Mutations in PARK7 are a rare cause of familial recessive Parkinson's disease (PD), but growing evidence suggests involvement of DJ ...

The hallmark of Parkinson's disease (PD) is the selective loss of dopamine neurons in the ventral midbrain. Although the cause of neurodegeneration in PD is unknown, a Mendelian inheritance pattern is observed in rare cases, indicating a genetic factor. Furthermore, pathological analyses of PD substantia nigra have correlated cellular oxidative stress and altered proteasomal function with PD. Homozygous mutations in DJ-1 were recently described in two families with autosomal recessive Parkinsonism, one of which is a large deletion that is likely to lead to loss of function. Here we show that embryonic stem cells deficient in DJ-1 display increased sensitivity to oxidative stress and proteasomal inhibition. The accumulation of reactive oxygen species in toxin-treated DJ-1-deficient cells initially appears normal, but these cells are unable to cope with the consequent damage that ultimately leads to apoptotic death. Furthermore, we find that dopamine neurons derived from in vitro-differentiated DJ-1-deficient embryonic stem cells display decreased survival and increased sensitivity to oxidative stress. These data are consistent with a protective role for DJ-1, and demonstrate the utility of genetically modified embryonic stem cell-derived neurons as cellular models of neuronal disorders.

Parkinson’s disease (PD; OMIM #168600) is a common neurodegenerative disorder characterised by bradykinesia, resting tremor, muscle rigidity, and postural instability. The pathological features include loss of dopaminergic neurones, in particular within the substantia nigra pars compacta, and eosinophilic, cytoplasmic inclusions termed Lewy bodies.1 Although rare, familial forms of parkinsonism provide a powerful tool to determine the molecular pathways perturbed in idiopathic PD.2,3 Three loci have been associated with autosomal recessive early onset parkinsonism (EO-PD): parkin ( PARK2 ),4 the as yet unidentified PARK6 ,5 and DJ-1 ( PARK7 ).6 Loss of Parkin function is the predominant genetic cause of EO-PD in Japanese, Northern European, North American, and North African populations. Homozygous and compound heterozygous parkin mutations account for approximately 49% of familial and 19% of sporadic EO-PD (with onset prior to 45 years).7–13 Although the gene has yet to be identified, linkage analysis of chromosome 1p36 interval suggests that PARK6 mutations may also account for numerous families with EO-PD across Europe.14 Most recently, mutations in DJ-1 ( PARK7 ) were identified in consanguineous Dutch and Italian kindreds affected with EO-PD. In the Dutch kindred, a 14 kb genomic deletion removed the promoter region and the first five exons (Δ1−5). In the Italian kindred, a highly conserved amino acid (leucine) was altered to proline (497 bp T→C; L166P). This substitution was predicted to disrupt protein folding and was demonstrated to affect cellular localisation.6 Both mutations showed complete segregation with disease in homozygous individuals, while heterozygous carriers were unaffected, suggesting that loss of function of DJ-1 is pathogenic. To date, DJ-1 Δ1–5 and 497 bp T→C (L166P) mutations have only been reported in the original Dutch and Italian families and in ethnically matched control individuals.6 There have been no reports on the frequency of DJ-1 …

The proper communication between gut and brain is pivotal for the maintenance of health and, dysregulation of the gut-brain axis can lead to several clinical disorders. In Parkinson’s disease (PD) 85% of all patients experienced constipation many years before showing any signs of motor phenotypes. For differential diagnosis and preventive treatment, there is an urgent need for the identification of biomarkers indicating early disease stages long before the disease phenotype manifests. DJ-1 is a chaperone protein involved in the protection against PD and genetic mutations in this protein have been shown to cause familial PD. However, how the deficiency of DJ-1 influences the risk of PD remains incompletely understood. In the present study, we provide evidence that DJ-1 is implicated in shaping the gut microbiome including; their metabolite production, inflammation and innate immune cells (ILCs) development. We revealed that deficiency of DJ-1 leads to a significant increase in two specific genera/species, namely Alistipes and Rikenella. In DJ-1 knock-out (DJ-1-/-) mice the production of fecal calprotectin and MCP-1 inflammatory proteins were elevated. Fecal and serum metabolic profile showed that malonate which influences the immune system was significantly more abundant in DJ-1−/− mice. DJ-1 appeared also to be involved in ILCs development. Further, inflammatory genes related to PD were augmented in the midbrain of DJ-1−/− mice. Our data suggest that metabolites and inflammation produced in the gut could be used as biomarkers for PD detection. Perhaps, these metabolites and inflammatory mediators could be involved in triggering inflammation resulting in PD pathology.

Bi-allelic mutations in the genes Parkin (PARK2), PINK1 (PARK6) and DJ-1 (PARK7) are established causes of autosomal recessive early-onset Parkinson’s Disease (EOPD). PINK1 mutations are the second commonest cause of EOPD. Specific mutations may be relatively common in certain populations because of a founder effect. Homozygous p.A217D PINK1 mutations were previously shown to cause EOPD in a large Sudanese kindred. Here we report the segregation of homozygous PINK1 p.A217D mutations in a family originating in Morocco with a history of parental consanguinity. From the clinical information available for the index case, the phenotype of mild, slowly-progressive Parkinsonism is consistent with previous reports of p.A217D disease and of PINK1 disease phenotype more generally. The reported features of early prominent lower-limb symptoms and gait disturbance with asymmetrical onset are more frequent among PINK1 disease cases. Together, reports of p.A217D in families of Moroccan and Sudanese origin suggest that p.A217D is a North African mutation due to a founder effect. Wider genetic analyses of EOPD in North Africa would be useful to estimate the prevalence of Parkinsonism caused by PINK1 p.A217D. In the absence of bi-allelic Parkin mutations, PINK1 mutations should be considered in cases with evidence of autosomal recessive inheritance of EOPD and presentation of atypical features such as early lower-limb symptoms and gait disturbance with asymmetrical onset, which appear to be common in Mendelian EOPD.

Neuronal Ca2+ sensor proteins (NCS) transduce changes in Ca2+ homeostasis into altered signaling and neuronal function. NCS-1 activity has emerged as important for neuronal viability and pathophysiology. The progressive degeneration of dopaminergic (DA) neurons, particularly within the Substantia nigra (SN), is the hallmark of Parkinson's disease (PD), causing its motor symptoms. The activity-related Ca2+ homeostasis of SN DA neurons, mitochondrial dysfunction, and metabolic stress promote neurodegeneration and PD. In contrast, NCS-1 in general has neuroprotective effects. The underlying mechanisms are unclear. We analyzed transcriptional changes in SN DA neurons upon NCS-1 loss by combining UV-laser microdissection and RT-qPCR-approaches to compare expression levels of a panel of PD and/or Ca2+-stress related genes from wildtype and NCS-1 KO mice. In NCS-1 KO, we detected significantly lower mRNA levels of mitochondrially coded ND1, a subunit of the respiratory chain, and of the neuron-specific enolase ENO2, a glycolytic enzyme. We also detected lower levels of the mitochondrial uncoupling proteins UCP4 and UCP5, the PARK7 gene product DJ-1, and the voltage-gated Ca2+ channel Cav2.3 in SN DA neurons from NCS-1 KO. Transcripts of other analyzed uncoupling proteins (UCPs), mitochondrial Ca2+ transporters, PARK genes, and ion channels were not altered. As Cav channels are linked to regulation of gene expression, metabolic stress and degeneration of SN DA neurons in PD, we analyzed Cav2.3 KO mice, to address if the transcriptional changes in NCS-1 KO were also present in Cav.2.3 KO, and thus probably correlated with lower Cav2.3 transcripts. However, in SN DA neurons from Cav2.3 KO mice, ND1 mRNA as well as genomic DNA levels were elevated, while ENO2, UCP4, UCP5, and DJ-1 transcript levels were not altered. In conclusion, our data indicate a possible novel function of NCS-1 in regulating gene transcription or stabilization of mRNAs in SN DA neurons. Although we do not provide functional data, our findings at the transcript level could point to impaired ATP production (lower ND1 and ENO2) and elevated metabolic stress (lower UCP4, UCP5, and DJ-1 levels) in SN DA neurons from NCS-1 KO mice. We speculate that NCS-1 is involved in stimulating ATP synthesis, while at the same time controlling mitochondrial metabolic stress, and in this way could protect SN DA neurons from degeneration.

Protein lipoxidation was assessed in the parietal cortex (PC), frontal cortex (FC), and cingulate gyrus (CG) in middle-aged and old-aged individuals with no clinical manifestations of cognitive impairment, in order to increase understanding of regional brain vulnerability to oxidative damage during aging. Twenty-five lipoxidized proteins were identified in all the three regions although with regional specificities, by using redox proteomics to detect target proteins of neuroketals (NKT) adduction. The number of cases with NKT-adducted proteins was higher in old-aged individuals but most oxidized proteins were already present in middle-aged individuals. Differences in vulnerability to oxidation were dependent on the sub-cellular localization, secondary structure, and external exposition of certain amino acids. Lipoxidized proteins included those involved in energy metabolism, cytoskeleton, proteostasis, neurotransmission and O2/CO2, and heme metabolism. Total NKT and soluble oligomer levels were estimated employing slot-blot, and these were compared between age groups. Oligomers increased with age in PC and FC; NKT significantly increased with age in FC, whereas total NKT and oligomer levels were not modified in CG, thus highlighting differences in brain regional vulnerability with age. Oligomers significantly correlated with NKT levels in the three cortical regions, suggesting that protein NKT adduction parallels soluble oligomer formation. This study was funded by the Seventh Framework Programme of the European Commission, grant agreement 278486: DEVELAGE, and by the Ministerio de Ciencia e Innovación, Instituto de Salud Carlos III – Fondos FEDER, a way to build Europe FIS grants PI14/00757 and PI14/00328. We wish to thank T. Yohannan for editorial help.

Purpose Growth factors and inflammatory and angiogenetic proteins are involved in the development of retinopathy of prematurity (ROP). However, no early biochemical markers are in clinical use to predict ROP. By performing cluster analysis of multiple biomarkers, we aimed to determine patient groups with high and low risk for developing ROP. Methods In total, 202 protein markers in plasma were quantified by proximity extension assay from 35 extremely preterm infants on day 2 of life. Infants were sorted in groups by automated two-dimensional hierarchical clustering of all biomarkers. ROP was classified as stages I to III with or without surgical treatment. Predictive biomarkers were evaluated by analysis of variance and detected differences by two-sided paired t-test with Bonferroni corrections for multiple comparisons. Results Differences in 39 biochemical markers divided infants without ROP into two control groups (control 1, n = 7; control 2, n = 5; P < 0.05). Sixty-six biochemical markers defined differences between the control groups (n = 13) and all ROP infants (n = 23; P < 0.05). PARK7, VIM, MPO, CD69, and NEMO were markedly increased in control 1 compared to all ROP infants (P < 0.001). Lower TNFRSF4 and higher HER2 and GAL appeared in infants with ROP as compared to control 1 and/or 2 (P < 0.05, respectively). Conclusions Our data suggest that early elevated levels of PARK7, VIM, MPO, CD69, and NEMO may be associated with lower risk of developing ROP. Lower levels of TNFRSF4 with higher levels of HER2 and GAL may predict ROP development. Translational Relevance Cluster analysis of early postnatal biomarkers may help to identify infants with low or high risk of developing ROP.